The loss of genomic stability in germ cells contributes to fertility and birth defects, and genomic disorders in human. Maintaining genomic stability requires accurate repair of DNA damage. A poorly- defined form of meiotic DNA double-strand break (DSB) repair is homologous recombination between sister chromatids. In humans, the mis-regulation of meiotic sister-chromatid recombination is thought to generate disease-associated rearrangements of the Y chromosome (Lange et al., Cell 138, 855 (2009)). A major barrier to studying this crucial repair process is the lack of experimental systems to specifically interrogate sister-chromatid recombination in meiosis, because there is frequent recombination occurring between homologous chromosomes. We are combining genetics and cytology in the roundworm Caenorhabditis elegans for the focused study of meiotic sister-chromatid recombination. We found the conserved SMC-5/6 protein complex functions specifically in meiotic sister-chromatid recombination. In the absence of SMC-5/6 function, inter-homolog recombination was unaffected, but inter-sister recombination was impaired leading to chromosome fragmentation. Due to a unique attribute of C. elegans chromosomes, the fragmentation defect did not result in mis-segregation. Consequently, we recovered viable offspring that showed gradual loss of germ cell immortality as later generations of offspring eventually became infertile. This project will utilize the C. elegans SMC-5/6 model to define the genetic pathway(s) that regulate meiotic inter-sister recombination, so that we can better explain how mis-regulation of this process might occur in humans. We identified candidate genes involved in this process based on the homologous recombination defects of the smc-5/6 mutants and the current models for homologous recombination repair. To complement this candidate gene approach, we will purify the SMC-5/6 protein complex to identify additional potentially novel candidate factors involved in inter-sister repair. These candidate genes will be tested for functions specifically in meiotic inter-sister recombination. Candidate genes will be systematically inactivated in genetic mutant backgrounds that only permit homolog-independent repair, and then specifically interrogated for homologous recombination repair using established cytological methods. For the second Aim of this project, we will directly define the types and frequency of mutations arising in the smc-5 and smc-6 mutants. We will perform a comprehensive analysis of DNA lesions accumulating at a defined genetic locus, using a powerful genetic system to identify de novo mutations in the unc-93 gene. This approach should allow us to identify any type of mutations. As a complementary approach we will perform genome-wide array Comparative Genomic Hybridization (aCGH) analysis to detect genomic duplication and deletion events. The combination of these two approaches will provide an unbiased test for whether the loss of SMC-5/6 function contributes to aberrant genomic rearrangements.